1. Field of the Invention
The present invention relates generally to the fields of cancer treatment and chemotherapy. More particularly, it concerns use of the protein kinase C (PKC) inhibitor chelerythrine to treat cancer in combination with DNA damaging chemicals to produce unexpectedly effective killing of tumor cells and tumor endothelial cells.
2. Description of Related Art
Certain cancer treatment methods, including chemotherapeutic therapy, involve damaging the DNA of the cancer cell. The cellular response to normal DNA damage includes activation of DNA repair, cell cycle arrest and lethality (Hall, 1988). For example, the induction of DNA double-strand breaks results in lethal chromosomal aberrations that include deletions, dicentrics, rings, and anaphase bridges (Hall, 1994).
The morphological characteristics of cells dying a mitotic death include, as in necrotic death, multi-nucleated giant cells, cell-cell fusions (Hall, 1994), as well as the loss of membrane integrity (Quintans et al., 1994; Maity et al., 1994; Harmon and Allan, 1988; Radford and Murphy, 1994). In contrast to necrotic death, morphological characteristics of apoptosis (Quintans et al., 1994; Maity et al., 1994; Harmon and Allan, 1988; Radford and Murphy, 1994) include activation of a genetic program that may be initiated by cytoplasmic or nuclear events which results in cytoplasmic blebbing, chromatin condensation, and DNA fragmentation (Jacobson et al., 1994; Raff et al., 1994).
Studies in tumor systems suggest that increasing the fraction of tumor cells undergoing apoptosis enhances tumor regression and tumor cures (Meyn et al., 1993; 1994; 1995; Martin and Green, 1994; Indap and Rao, 1995; Dewey et al., 1995; Lowe et al., 1993b; Stephens et al., 1991; 1993; Chmura et al., 1997). Agents which damage DNA, interfere with DNA repair, or alter cell-cycle checkpoints have been employed in human studies to modify tumor response with limited clinical success (Hall, 1988; Vokes and Weichselbaum, 1990; Rosenthal et al., 1995). Chelerythrine chloride (Herbert et al., 1990) and calphostin C (Kobayashi et al., 1989b), inhibitors of protein kinase C (PKC) isoforms, also induce apoptosis and ceramide production through the activation of a neutral sphingomyelinase (Chmura et al., 1996a; Chmura et al., 1996b). The protein kinase C family of serine/threonine kinases is comprised of at least 13 related isoforms (Magnuson et al., 1994) with differing sensitivity to calcium and lipid activators.
Little information is available concerning the relationship between PKC inhibitors and the induction of programmed cell death in human tumor cells, and the results described in existing reports are inconsistent. For example, the potent, but nonspecific, PKC inhibitor staurosporine has been reported both to antagonize (Cotter et al., 1992) and to initiate apoptosis in HL-60 cells (Bertrand et al., 1993); similarly conflicting reports of the action of the inhibitor H7 have also appeared (Ojeda et al., 1990; Forbes et al., 1992). Detailed comparisons of the concentration-response relationships of different PKC inhibitors in the modulation of apoptosis are generally lacking. Jarvis et al. (1994) demonstrate that, while the effects of these agents are variable and highly dependent upon concentration, transient exposure of HL-60 cells to a subset of PKC inhibitors, in particular chelerythrine, unambiguously induces apoptotic DNA fragmentation and cell death in HL-60 cells, and that acute (i.e., 6 h) exposure to chelerythrine is sufficient to induce apoptosis in the human myeloid leukemia cell line HL-60. In addition, in vitro treatment of certain cells with inhibitors of PKC and other serine-threonine kinases increases IR mediated killing through undefined mechanisms (Hallahan et al., 1992).
Recent investigations indicate that signaling events following cellular exposure to tumor necrosis factor alpha (TNFxcex1), Fas ligand, IgM cross-linking, irradiation and other DNA damaging agents may trigger apoptosis via the hydrolysis of membrane sphingomyelin generating ceramide (Quintans et al., 1994; Nagata and Golstein, 1995; Dressler et al., 1992). Activation of PKC by phorbol esters or growth factors opposes ceramide-induced apoptosis and indirect evidence suggests that PKC activation may limit ceramide production (Fuks et al., 1994; Haimovitz-Friedman et al., 1994a; Haimovitz-Friedman et al., 1994b). In tumor endothelial cells, one potential action of ceramide and its metabolite sphingosine is to prevent activation of specific PKC isoforms (Chmura et al., 1996b; Jones and Murray, 1995; Kolesnick, 1989; Ohta et al., 1994). Taken together, these studies suggest that PKC activation may oppose the actions of ceramide production in the apoptotic pathway in tumor cells and tumor endothelial cells.
Though it is clear that PKC pathways play a key role in the control of apoptosis by tumor cells, how this is accomplished remains obscure. Thus, there remains a present need to develop new and improved treatments which take advantage of these pathways.
The invention provides a method for inhibiting growth of a tumor cell comprising contacting the tumor cell and/or the tumor endothelial cell with chelerythrine and contacting the tumor cell with DNA damaging agent, wherein the dose of the chelerythrine, when combined with the dose of the DNA damaging agent, is effective to inhibit growth of the tumor cell.
The invention further provides a method for inhibiting the growth of a cell comprising contacting the cell with chelerythrine and a chemotherapeutic DNA damaging agent, wherein the dose of chelerythrine, when combined with the dose of the DNA damaging agent, is effective to inhibit growth of the cell. In one embodiment of the method, chelerythrine is contacted with the cell prior to contacting the cell with the DNA damaging agent. In another embodiment of the method, the DNA damaging agent is contacted with the cell prior to contacting the cell with chelerythrine.
In a further embodiment of the method, the cell is a cancer cell. In a further aspect of the invention, the cancer cell is a bladder cancer cell, a blood cancer, a breast cancer cell, a lung cancer cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a pancreatic cancer cell, a stomach cancer cell, a testicular cancer cell, a brain cancer cell, an ovarian cancer cell, a lymphatic cancer cell, a skin cancer cell, a brain cancer cell, a bone cancer cell, or tumor of soft tissue. In yet another aspect of the invention, the cell is located in a human subject.
In one embodiment of the invention, chelerythrine is administered by direct intratumoral injection. In another embodiment of the invention, chelerythrine is administered by injection into tumor vasculature. In a further embodiment of the invention, the DNA damaging agent is from a group consisting of doxorubicin, daunorubicin, dactinomycin, mitoxantrone, cisplatin, procarbazine, mitomycin, carboplatin, bleomycin, etoposide, teniposide, mechlroethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, ifosfamide, melphalan, hexamethylmelamine, thiopeta, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, hydrogen peroxide, nitrosurea, plicomycin, tamoxifen, taxol, transplatinum, vincristin, vinblastin and methotrexate.
In one aspect of the invention, the cell is contacted with chelerythrine a second time. In another aspect of the invention, the cell is contacted with the DNA damaging agent a second time. In one embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 4 days. In another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 3 days. In yet another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 2 days. In still another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 1 day. In still yet another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 12 hours. In still another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 6 hours. In yet another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 5 hours. In still other embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 4 hours. In yet other embodiments of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 3 hours. In still another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 2 hours. In another embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 1 hour. In a further embodiment of the invention, chelerythrine and the DNA damaging agent are contacted with the cell at the same time. Thus, in various embodiments of the invention, chelerythrine and the DNA damaging agent are contacted with the cell within about 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 5.5 hours, 5 hours, 4.5 hours, 4 hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 0.5 hour or at the same time.
In one aspect of the invention, the chelerythrine and the DNA damaging agent are contacted with the cell following tumor resection. In one embodiment of the invention, the tumor resection occurs prior to the contacting. In one aspect of the invention, the contacting comprises treating the resected tumor bed with chelerythrine and the DNA damaging agent. In another embodiment of the invention the tumor resection occurs after the contacting. In a further embodiment of the invention, the contacting occurs both before and after the tumor resection.
In one embodiment of the invention, the dose of chelerythrine is about 0.5 mg/kg to about 10 mg/kg. In another embodiment of the invention, the dose of chelerythrine is about 1 mg/kg to about 4 mg/kg. However, one of skill in the art will recognize that the dose of chelerythrine administered will depend on the animal or human being treated and will be decided by factors such as, but not limited to, the age and weight of animal or patient and may vary by a log or more of the values described above. The skilled artisan is directed to xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d 15th Edition, chapter 33, in particular pages 624-652. The skilled artisan will further recognize that some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
The invention further provides a method of killing a cell comprising contacting the tumor cell with chelerythrine and a chemotherapeutic DNA damaging agent, wherein the dose of the chelerythrine, when combined with the dose of the DNA damaging agent, is effective to kill the tumor cell. In one embodiment of the invention, a method of treating cancer in a human patient comprising administering chelerythrine and a chemotherapeutic DNA damaging agent to the human patient, wherein the dose of the chelerythrine, when combined with the dose of the DNA damaging agent, is effective to treat the cancer. In a further embodiment of the invention, a method of potentiating the effect of a chemotherapeutic DNA damaging agent on a tumor cell and/or a tumor endothelial cell comprising contacting the tumor cell and/or a tumor endothelial cell with chelerythrine and then contacting the tumor cell and/or a tumor endothelial cell with the DNA damaging agent.
As defined herein, treatment of a cancer, tumor, tumor cell, tumor endothelial cell, cancer cell, tissue derived from a tumor or cancerous tissue refers to any improvement over the untreated state which includes, but is not limited to, stabilization, remission, regression, shrinkage or decreased volume of the cancer, tumor, tissue or cell.